Following activation by antigen, co-stimulation and inflammation, naive CD8+ T cells initiate a differentiation program resulting in massive changes in gene expression and cell function, which leads to the formation of effector and memory T cells (Doering et al. (2012) Immunity 37:1130-1144). This differentiation program is critical for the development of effective tumor immunity (Galon et al. (2006) Science 313:1960-1964) and the control of pathogens (Wong and Pamer (2003) Annu. Rev. Immunol. 21:29-70). However, the developing effector CD8+ T cells population is strikingly heterogeneous (Arsenio et al. (2014) Nat. Immunol. 15:365-372), and several phenotypically distinct subpopulations of T cells exist within the effector pool that have different lineage potential and function (Joshi et al. (2007) Immunity 27:281-295; Sarkar et al. (2008) J. Exp. Med. 205:625-640). Thus, CD8+ effector T cells face complex lineage choices during differentiation.
Although the development of effector CD8+ T cells occurs over a period of days (Kaech et al. (2002) (Cell 111:837-851), early events in the life-history of T cells are critical in determining the fate of T cells (Kaech and Ahmed (2001) Nat. Immunol. 2:415-422; Mercado et al. (2000) J. Immunol. 165:6833-6839; van Stipdonk et al. (2001) Nat. Immunol. 2:423-429; van Stipdonk et al. (2003) Nat. Immunol. 4:361-365; Wong and Pamer (2001) J. Immunol. 166:5864-5868). For instance, asymmetric segregation of cell contents during the first cell division after encounter with antigen can profoundly influence the ultimate differentiation state of effector cells (Chang et al. (20017) Science 315:1687-1691), suggesting that investigating the events that occur in the hours following antigen encounter will be essential to define the mechanisms that regulate the fate of effector CD8+ T cells.
Effector differentiation is regulated by a set of transcription factors (TFs) including T-bet (Intlekofer et al. (20015) Nat. Immunol. 6:1236-1244), Eomes (Pearce et al. (2003) Science 302:1041-1043), Blimp1 (Rutishauser et al. (2009) Immunity 31:296-308; Shin et al. (2009) Immunity 31:309-320), Id2 (Cannarile et al. (2006) Nat. Immunol. 7:1317-1325), and Runx3 (Cruz-Guilloty et al. (2009) J. Exp. Med. 206:51-59). It has recently been shown that the AP-1 family TF, BATF, is absolutely required for effector CD8+ T cell differentiation and coordinates the program of gene expression essential for this process (Kurachi et al. (2014) Nat. Immunol. 15:373-383). Thus, many TFs that are expressed immediately after stimulation may play a role in specifying the fate of developing effector cells from the earliest point in differentiation.
The role of specific TFs in regulating CD8+ T cell effector differentiation has been investigated using germline or conditional KOs. However, these approaches are limited to studying a small number of candidate genes (Kaech and Cui (2012) Nat. Rev. Immunol. 12:749-761). In contrast, perturbing genes with RNAi could in principle permit many more candidate regulators to be studied in parallel (Amit et al. (2009) Science 326:257-263). However, techniques to deliver shRNAs to T cells are limited by the need to stimulate cells to divide using TCR cross-linking (Yang et al. (2012). J. Exp. Med. 209:1655-1670), infection (Joshi et al. (2007) Immunity 27:281-295; Araki et al. (2009) Nature 460:108-112), or cytokine stimulation (Zhou et al. (2014) Nature 506:52-57) in order to achieve meaningful frequencies of transduction with viral vectors encoding shRNA constructs. This strategy of activating T cells in order to deliver shRNAs raises a concern that this activation could profoundly alter the cell at a critical phase of time when even subtle perturbations of TFs can profoundly influence T cell fate (Chang et al. (2007) Science 315:1687-1691).
Thus, new approaches, compositions, methods, and systems are required to perturb gene expression in many cell types, including immune and hematopoietic cell types, in a manner that preserves natural cellular differentiation.